Neutrophils (polymorphonuclear leukocytes) and macrophages play a major role in the body's defense against harmful foreign pathogens. In the course of serving this function, phagocytes must respond to a variety of inflammatory stimulants, including N-formylated peptides, complement component C5a, leukotriene B.sub.4 platelet-activating factor, and interleukin 8 (see, e.g., Becker, J. Leukocyte Biol. 47: 378-389 (1990)).
Phagocytes have specific cell surface receptors for these factors that are coupled to the intracellular environment through oligomeric GTP-binding proteins, which are also known as G proteins. These transducing elements are found in all cells and couple ligand-activated receptors to the intracellular second messenger cascades (Birnbaumer, et al., CRC Crit. Rev. Biochem. Mol. Biol. 25: 225-244 (1990)). Protein purification and molecular cloning have revealed the existence of multiple additional G proteins that bear homology with the ras oncogene products (Der, in Oncogenes, Benz & Liu (eds), Kluweer Academic, Boston, Mass., 1989, pp. 73-119; Hall, Science 249: 635-640 (1990)). These smaller monomeric proteins are referred to herein as low molecular weight GTP-binding proteins (LMWGs). Recent findings have implicated the LMWGs in a variety of neutrophil activities.
One of these activities is the eradication of disease-causing organisms. For example, the phagocytic cell NADPH oxidase generates superoxide anion (O.sub.2.sup.-) as a means to destroy ingested microorganisms. Its importance in bacterial killing is evidenced by the chronic infections and even death observed in patients with severe neutropenia, chronic granulomatous disease, and other disorders of neutrophil function. However, the inappropriate or excessive formation of O.sub.2.sup.- and its byproducts can both initiate and exacerbate inflammation. Inflammatory diseases and/or secondary inflammation resulting from a primary disorder are serious health problems. Therefore, the development of means to intervene in this process in a specific manner is of great therapeutic interest.
Human neutrophils and other phagocytic cells undergo a respiratory burst in which superoxide anion (O.sub.2.sup.-) and its metabolites--hydrogen peroxide (H.sub.2 O.sub.2), hypochlorous and other hypohalous acids, and hydroxyl radical (OH)--are produced as a means of destroying ingested microorganisms. The significance of the respiratory burst in host defense is made evident by the recurrent and life-threatening infections that occur in patients with chronic granulomatous disease (CGD) in whose phagocytes the burst does not occur. It is known that CGD results from genetic defects in any one of four known protein components of the NADPH oxidase enzyme responsible for generating O.sub.2.sup.- (see J. T. Curnutte, in Phagocytic Defects II: Abnormalities of the Respiratory Burst (vol. 2), J. T. Curnutte (ed.), Saunders, Philadelphia, pp. 241-252 (1988)). Studies with various cell-free oxidase activation systems (Curnutte, et al., J. Biol. Chem. 262: 450 (1987); Bromberg, et al., J. Biol. Chem. 220: 13539 (1985)) and cellular material from CGD patients have helped to determine the identity and function of the components of the NADPH oxidase. The oxidase is composed of membrane-bound proteins that include cytochrome bass and possibly a 45 kD flavoprotein, as well as cytosolic components, of which two have been characterized: p47.sub.[phox] and p67.sub.[phox] (Clark, J. Infect. Dis. 161: 1140 (1990); Smith and Curnutte, Blood 77: 673 (1991)). The NADPH oxidase is activated at least in part by the association of these components into a membrane-bound complex that can transfer electrons from NADPH to molecular oxygen, generating O.sub.2.sup.- (Clark, Id.; Smith and Curnutte, Id.).
GTP-binding proteins have recently been implicated in the regulation of NADPH oxidase activity. GTP appears to be required for oxidase activation in cell-free systems and GDP analogs inhibit this activation. (See, e.g., Gabig, et al., J. Biol. Chem. 262: 1685 (1987); Seifert, et al., FEBS Lett. 205: 161 (1986); Uhlinger, et al., J. Biol. Chem. 266: 20990-997 (1991); Pereri, et al., PNAS USA 89: 2494-98 (1992).) It has also been observed that inhibition of protein isoprenylation decreases the rate of O.sub.2.sup.- generation. (See, e.g., Knaus, et al., Science 254: 1512-1515 (1991); Bokoch and Prossnitz, J. Clin. Invest. 89: 402-408 (1992)); and Chuang, et al., J. Biol. Chem. 268: 775-778 (1993).)
NADPH oxidase can be activated in cell-free systems containing cytosol and membranes from unstimulated phagocytes by the addition of an anionic amphiphile such as arachidonate or sodium dodecyl sulfate (SDS). (See, e.g., Curnutte, J. Clin. Invest. 75: 1740-1743 (1985); Bromberg and Pick, Cell. Immunol. 88: 213-221 (1984); McPhail, et al., J. Clin. Invest. 75: 1735-1739 (1985).) Several reports have shown that GTP or one of its non-hydrolyzable analogs (e.g., guanosine 5'-o-(3-thiotriphosphate) or GTP.gamma.S) may cause a two- to four-fold enhancement in the rate of O.sub.2.sup.- generation by these systems (Gabig, et al., J. Biol. Chem. 262: 1685-1690 (1987); Seifert, et al., FEBS Lett. 205: 161-165 (1986)). More recently, we have demonstrated that there is an absolute requirement for GTP (or GTP.gamma.S) in the cell-free system (Uhlinger, et al., J. Biol. Chem. 266: 20990-20997 (1991); Peveri, et al., PNAS USA 89: 2494-2498 (1992)). Moreover, the NADPH oxidase activity of differentiated HL-60 cells is dependent upon prenylation of a cytosolic component (Bokoch and Prossnitz, J. Clin. Invest. 89: 402-408 (1992)). Taken together, this evidence is strongly indicative of a role for a GTP-binding protein in NADPH oxidase activation.
Two very closely related members of the Rho family of Ras-like LMWGs have recently been implicated in the regulation of NADPH oxidase. We purified Rac2 from human neutrophil cytosol on the basis of its ability both to bind GTP.gamma.S and stimulate O.sub.2.sup.- generation in the presence of a suboptimal amount of neutrophil cytosol (Knaus, et al., Science 254: 1512-1515 (1991); Knaus, et al., J. Biol. Chem. 267: 23575-23582 (1992)). In similar experiments using guinea pig peritoneal macrophages, Rac1 was purified in an oxidase-enhancing complex (termed .delta.1) with Rho GDP dissociation inhibitor (RhoGDI). (See, e.g., Abo, et al., Nature 353: 668-670 (1991).) Subsequently, Mizuno, et al., J. Biol. Chem. 267: 10215-10218 (1992) also purified Rac2 from differentiated HL-60 cells, a human myeloid cell line, and confirmed that it enhances NADPH oxidase activity in a cell-free assay. A different approach was taken by Dorseuil and colleagues (J. Biol. Chem. 267: 20540-20542 (1992)), who used Epstein-Barr virus-transformed B lymphocytes that produce O.sub.2.sup.- by an NADPH oxidase system similar (and perhaps identical) to the one in phagocytic cells. They showed that rac antisense (but not sense) oligonucleotides decreased the Rac protein content of the cells and inhibited O.sub.2.sup.- generation in a dose-dependent manner, thus confirming the physiological role of Rac proteins in the regulation of NADPH oxidase activity. Rac1 and Rac2 are 92% identical and both undergo post-translational modification by the addition of a 20-carbon geranylgeranyl group to the cysteine of the carboxy-terminal CAAX box (Kinsella, et al., J. Biol. Chem. 266: 9786-9794 (1991)). Rac1 is expressed in a wide variety of cell types, whereas Rac2 is apparently restricted to cells of myeloid and lymphoid origin (Didsbury, et al., J. Biol. Chem. 264: 16378-16382 (1989); Reibel, et al., Biochem. Biophys. Res. Commun. 175: 451-458 (1991)).